Thesis (doctoral)--Lund University, 1989. / Added t.p. with thesis statement inserted.

In Drosophila, the insulin signaling pathway is at the interface between dietary conditions and control of growth and development, reproduction, stress responses and life span. Eight insulin like peptides (Dilp1-8), an insulin tyrosine kinase receptor (dInR) and its downstream components, as well as a relaxin-like receptor type (Lgr3) form the core of this signaling. Here we showed that the dInR mediates post-mitotic cell growth specifically in about 300 peptidergic neurons expressing the basic helix loop helix (bHLH) transcription factor Dimmed (Paper I).  Overexpression of dInR in Dimm positive neurons leads to increased size of cell body, Golgi apparatus and nucleus, whereas dInR knockdown causes an opposite effect. Manipulation of downstream components of insulin signaling induces similar changes in Dimm positive neurons. This mechanism is nutrient dependent. In Paper II, we further investigate the relation between Dimmed and dInR for regulation of cell growth. Coexpressing Dimm and dInR in a range of Dimm negative neurons results in increased cell size in both larval and adult stages. We provide further evidence that dInR regulates cell growth in a Dimm dependent manner and that DILP6 from glia cells is involved in this regulation. In addition, we find that Dimm alone is capable of triggering cell growth in certain neuron types at different developmental stages. Furthermore, ectopic Dimm alone can block apoptosis.  Dimm is a known master regulator of peptidergic cell fate. In paper III we find that ectopic expression of Dimm in Dimm negative motor neurons results in transformation the neurons towards a neuroendocrine phenotype. They acquire enlarged axon terminations and boutons, lose both pre- and postsynaptic markers, and display diminished levels of wingless and its receptor dFrizzled. Furthermore they show increased expression of several Dimm targets. Finally, combined ectopic Dimm and dInR expression gives rise to stronger phenotypes. In paper IV we studied another DILP possibly involved in growth regulation, the under-investigated DILP1. We generated Dilp1-Gal4 lines and anti DILP1 antibodies and found that DILP1 is transiently expressed in brain insulin producing cells (IPCs) from pupal stages to newly hatched adult flies. Diapausing virgin female flies display a high expression level of dilp1/DILP1 over at least 9 weeks of adult life. DILP1 expression is also correlated with the persistence of larval/pupal fat body and its expression is regulated by other DILPs and short neuropeptide F (sNPF). Flies mutant in dilp1 display increased food intake, but decreased stress resistance and life span. We found no obvious role of DILP1 in growth regulation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>

ABSTRACT Insulin Dynamic Measures and Weight Change By Noreen Kloc B.S. Computer Information Technology, Purdue University December 7, 2015 INTRODUCTION: Weight gain and obesity are risk factors for insulin resistance that can lead to type 2 diabetes and cardiovascular disease; however, there is a complicated interplay between insulin sensitivity (SI), fasting insulin, acute insulin response (AIR), and disposition index (DI) and the relationship of these dynamic measures with weight change is not well understood. AIM: The aim of this study was to investigate the relationships between insulin dynamic measures, SI, fasting insulin, AIR, and DI, with weight change during a 5-years follow-up period in the multi-ethnic cohort of the Insulin Resistance Atherosclerosis Study (IRAS). METHODS: Data on 879 men and women of Hispanic, non-Hispanic White, and African-American race/ethnicity aged 40-69 years were obtained at baseline (1992-1994) and at 5 year follow-up. Crude associations between the insulin dynamic measures and weight change were evaluated using Kruskal-Wallis test and the relationships between log-transformed insulin-related variables were examined using Spearman rank-order analysis. Multivariate regression models evaluated associations of interest adjusted for age, sex, ethnicity, and diabetes status in a time-dependent manner using mixed models. RESULTS: Insulin sensitivity SI inversely coevolves with weight, i.e. greater weight is predicted by lower SI at any time point. To answer the question whether SI is the cause or a consequence of weight change, we examined the associations with the baseline values and a change in SI. In this model, both the baseline SI and change in SI were inversely correlated with weight gain. A similar approach showed that baseline values and change in fasting insulin were directly associated with weight gain. Weight change over time was associated with AIR, i.e. increases in AIR and greater AIR at baseline predicted weight gain. We did not find strong relationships between DI and weight change. DISCUSSION: These results suggest that insulin sensitivity and insulin secretion can modulate weight in a non-diabetic population.

No description available.

Type 2 diabetes or adult onset diabetes, has been a global epidemic for the past two decades, and the number of new cases accelerates every year. Insulin resistance is one of the major factors behind this, wherein the insulin receptor, which signals to regulate glucose levels, based on the hormone insulin, loses its sensitivity. Obesity is one other major concern which is caused due to the improper balance between the caloric intake and the energy utilized. Gastric bypass surgeries (GBP) are performed to avert obesity. However, a beneficial side-effect is that the state of insulin resistance is reset to near baseline levels within a few days after the procedure. The reason behind this remains unexplained, with possible humoral effects, hypothesized to occur after the bariatric procedure. In this work, high-five insect cell line was utilized to recombinantly produce full length insulin receptors (IR). However commercially sourced IR ectodomains (eIR – soluble version of the full length IR with the completely extracellular α subunits along with extracellular and transmembrane regions of the β subunit), were used to study the interaction. Measuring the kinetics of IR-insulin interactions is critical to improving our understanding of this disease. In this study, a multiplex surface plasmon resonance (SPR) assay was developed for studying the interaction between insulin and the eIR. A scaffold approach was used in which anti-insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR capture. The multiplex SPR system (ProteOn XPR36TM, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. The main approach was to replicate the physiological IR-insulin interaction using this assay. It was also observed that insulin at higher concentrations tend to form dimers and hexamers in solution. This was tested using size exclusion chromatography analysis and proved to be true. Therefore an alternative analyte with the similar binding properties and affinity of insulin and at the same time with reduced self- association characteristics was explored. Lispro, the analogue of insulin with reduced self-association properties (generated by swapping of residue 28 and 29 with Lys and Pro respectively) was finally used to study the interaction with eIR. Interactions between recombinant human insulin with eIR-A (A isoform of the insulin receptor ectodomain) followed a two-site binding pattern (consistent with the literature), with a high-affinity site (dissociation constant KD1 = 38.1 ± 0.9 nM) and a low-affinity site (KD2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants KD1 =73.2 ± 1.8 nM and KD2 =148.9 ± 6.1 nM, but the fit to kinetic data was improved when conformational change factor was included in which the high-affinity site was converted to the low-affinity site. Kinetics of interaction of insulin with eIR-A and eIR-B isoforms were then compared. eIR-A bound insulin with apparently higher affinity (with both the binding sites) when compared with eIR-B. This was again consistent with literature that IR-A had two-fold higher affinity for binding insulin than IR-B. The assay was further extended to study the effect of external factors such as glucose, visfatin on this interaction. Glucose (the main biomolecule which is regulated by the IR-insulin interaction) was tested, if it had any direct effect on the interaction. It was observed that glucose did not have any effect on eIR-insulin interactions. Visfatin, an adipocytokine which has been highly debated in literature for its insulin mimetic effects and IR binding properties, was then tested. The standard assay did not provide much insights as the reference channel immobilized with 83-7 monoclonal antibody to the receptor had much affinity for visfatin, leading to non-specific binding and negative responses. Therefore, in an alternative methodology was used - visfatin, Lispro and insulin were immobilized on separate channels along with bovine serum albumin immobilized on reference channel and eIR isoforms used as analyte to study the effect of visfatin on IR. This study showed that visfatin, a higher molecular weight protein compared to insulin, bound both the eIR isoforms. This is consistent with literature that visfatin binds IR at a site distinct from insulin, but the assay described here could not confirm the fact that it mimicked the signalling carried out by IR-insulin binding. Further studies are required to interpret the kinetics of visfatin-eIR interaction. To my knowledge, this is the first SPR assay developed to study eIR-insulin interactions in real-time. This could potentially be extended to study the interaction of insulin with full length insulin receptors and the effect of humoral and other external factors on the interaction, without the need for insulin labelling.

No description available.

Class of 2009 Abstract / OBJECTIVES: The objective of this study was to investigate the effects of temperature excursions on insulin during standard shipping from mail-order pharmacies. METHODS: Twelve vials of insulin (six of regular and six of neutral protamine hagedorn (NPH)) were sampled at baseline and then the six experimental vials (three regular and three NPH) were shipped through the mail system from a Tucson, Arizona post office to a Tucson, Arizona residence. The other six vials were used as controls and left in a refrigerator at 5°C. Samples were taken daily and then measured for degradation using high performance liquid chromatography (HPLC). Also, samples from control vials were put in a lab oven at a constant temperature of 48°C and analyzed at Day 0 and Day 2. RESULTS: Temperatures spiked daily to near or over 50°C with a peak of 51.5°C. The low temperature never dropped under 21°C. The area under the curve (AUC) for each individual sample drawn was used to calculate a percentage of its original concentration with Day 1 set as 100%. On Day 6, both experimental vials and control vials had similar results and were within 10% of the original concentrations measured. In the oven, NPH samples that were heated for two days lost about 4% of its concentration while the regular insulin sample lost 14%. Particle sizing data of regular insulin heated in the oven was consistent with this HPLC data, and showed significant shifts in peak position. CONCLUSIONS: Insulin appears to maintain its stability after being shipped through the mail and remaining in a mailbox for an additional five days at high summer temperatures in Arizona. However, when exposed to constant high temperatures in a laboratory oven, heat appears to affect its stability.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg / Throughout the world, many traditional plant treatments for diabetes exist and therein lies a hidden wealth of potentially useful natural products for diabetes contrl. Despite this, few traditional antidiabetic plants have received scientific or medical scrutiny, and the World Health Organisation ( 1980 ) recommended accordingly that this area warrants further evaluation. / IT2018

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (M.A.)--University of North Carolina at Chapel Hill, 2004. / Includes bibliographical references (leaves 33-36). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.